U.S. patent application number 09/904603 was filed with the patent office on 2003-05-29 for novel microtubule-associated protein.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Goli, Surya K., Hillman, Jennifer L..
Application Number | 20030099612 09/904603 |
Document ID | / |
Family ID | 25190714 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030099612 |
Kind Code |
A1 |
Hillman, Jennifer L. ; et
al. |
May 29, 2003 |
Novel microtubule-associated protein
Abstract
The present invention provides a human microtubule-associated
protein (hLC3) and the polynucleotides which identify and encode
hLC3. The invention also provides genetically engineered expression
vectors, host cells, and a method for producing hLC3. The invention
also provides hLC3 as well as agonists, antibodies, and antagonists
of hLC3, and the use thereof for the prevention and treatment of
diseases associated with the expression of hLC3. Additionally, the
invention provides for the use of antisense molecules to
polynucleotides encoding hLC3 for the treatment of diseases
associated with the expression of hLC3. The invention also provides
diagnostic assays which utilize the polynucleotide, or fragments or
the complement thereof, and antibodies specifically binding
hLC3.
Inventors: |
Hillman, Jennifer L.;
(Mountain View, CA) ; Goli, Surya K.; (Sunnyvale,
CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
25190714 |
Appl. No.: |
09/904603 |
Filed: |
July 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09904603 |
Jul 12, 2001 |
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09199838 |
Nov 24, 1998 |
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6280733 |
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09199838 |
Nov 24, 1998 |
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08805117 |
Feb 24, 1997 |
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5955312 |
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Current U.S.
Class: |
424/93.2 ;
435/199; 435/252.3; 435/320.1; 435/325; 435/6.11; 435/69.1;
530/358; 536/23.2; 800/8 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/47 20130101 |
Class at
Publication: |
424/93.2 ; 435/6;
435/69.1; 435/199; 435/320.1; 435/325; 530/358; 536/23.2; 800/8;
435/252.3 |
International
Class: |
C12Q 001/68; A01K
067/00; C07H 021/04; A61K 048/00; C12N 009/22; C12P 021/02; C12N
005/06; C12N 001/21 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1,
b) a polypeptide comprising a naturally occurring amino acid
sequence at least 90% identical to the amino acid sequence of SEQ
ID NO:1, c) a fragment of a polypeptide having the amino acid
sequence of SEQ ID NO:1, wherein said fragment binds to
microtubules and d) an immunogenic fragment of a polypeptide having
the amino acid sequence of SEQ ID NO:1.
2. An isolated polypeptide of claim 1 comprising the amino acid
sequence of SEQ ID NO:1.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising the
polynucleotide sequence of SEQ ID NO:2.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide encoded by the
polynucleotide of claim 3, the method comprising: a) culturing a
cell under conditions suitable for expression of the polypeptide,
wherein said cell is transformed with a recombinant polynucleotide,
and said recombinant polynucleotide comprises a promoter sequence
operably linked to the polynucleotide of claim 3, and b) recovering
the polypeptide so expressed.
10. An isolated antibody which specifically binds to a polypeptide
of claim 1.
11. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising the polynucleotide sequence of
SEQ ID NO:2, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to the
polynucleotide sequence of SEQ ID NO:2, c) a polynucleotide
complementary to a polynucleotide of a), d) a polynucleotide
complementary to a polynucleotide of b), and e) an RNA equivalent
of a)-d).
12. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 11.
13. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
14. A method of claim 13, wherein the probe comprises at least 60
contiguous nucleotides.
15. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
16. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
17. A composition of claim 16, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:1.
18. A method for treating a disease or condition associated with
decreased expression of functional hLC3, comprising administering
to a patient in need of such treatment the composition of claim
16.
19. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
20. A composition comprising an agonist compound identified by a
method of claim 19 and a pharmaceutically acceptable excipient.
21. A method for treating a disease or condition associated with
decreased expression of functional hLC3, comprising administering
to a patient in need of such treatment a composition of claim
20.
22. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
23. A composition comprising an antagonist compound identified by a
method of claim 22 and a pharmaceutically acceptable excipient.
24. A method for treating a disease or condition associated with
overexpression of functional hLC3, comprising administering to a
patient in need of such treatment a composition of claim 23.
25. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, said method comprising the steps of: a)
combining the polypeptide of claim 1 with at least one test
compound under suitable conditions, and b) detecting binding of the
polypeptide of claim 1 to the test compound, thereby identifying a
compound that specifically binds to the polypeptide of claim 1.
26. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
27. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
28. A method for assessing toxicity of a test compound, said method
comprising: a) treating a biological sample containing nucleic
acids with the test compound; b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 11 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 11 or fragment thereof; c)
quantifying the amount of hybridization complex; and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
29. A diagnostic test for a condition or disease associated with
the expression of hLC3 in a biological sample comprising the steps
of: a) combining the biological sample with an antibody of claim
10, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex; and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
30. The antibody of claim 10, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
31. A composition comprising an antibody of claim 10 and an
acceptable excipient.
32. A method of diagnosing a condition or disease associated with
the expression of hLC3 in a subject, comprising administering to
said subject an effective amount of the composition of claim
31.
33. A composition of claim 31, wherein the antibody is labeled.
34. A method of diagnosing a condition or disease associated with
the expression of hLC3 in a subject, comprising administering to
said subject an effective amount of the composition of claim
33.
35. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 10 comprising: a) immunizing
an animal with a polypeptide having the amino acid sequence of SEQ
ID NO:1, or an immunogenic fragment thereof, under conditions to
elicit an antibody response; b) isolating antibodies from said
animal; and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide having the amino acid sequence of SEQ
ID NO:1.
36. An antibody produced by a method of claim 35.
37. A composition comprising the antibody of claim 36 and a
suitable carrier.
38. A method of making a monoclonal antibody with the specificity
of the antibody of claim 10 comprising: a) immunizing an animal
with a polypeptide having the amino acid sequence of SEQ ID NO:1,
or an immunogenic fragment thereof, under conditions to elicit an
antibody response; b) isolating antibody producing cells from the
animal; c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells; d)
culturing the hybridoma cells; and e) isolating from the culture
monoclonal antibody which binds specifically to a polypeptide
having the amino acid sequence of SEQ ID NO:1.
39. A monoclonal antibody produced by a method of claim 38.
40. A composition comprising the antibody of claim 39 and a
suitable carrier.
41. The antibody of claim 10, wherein the antibody is produced by
screening a Fab expression library.
42. The antibody of claim 10, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
43. A method for detecting a polypeptide having the amino acid
sequence of SEQ ID NO:1 in a sample, comprising the steps of: a)
incubating the antibody of claim 10 with a sample under conditions
to allow specific binding of the antibody and the polypeptide; and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide having the amino acid sequence of SEQ
ID NO:1 in the sample.
44. A method of purifying a polypeptide having the amino acid
sequence of SEQ ID NO:1 from a sample, the method comprising: a)
incubating the antibody of claim 10 with a sample under conditions
to allow specific binding of the antibody and the polypeptide; and
b) separating the antibody from the sample and obtaining the
purified polypeptide having the amino acid sequence of SEQ ID NO:1.
Description
[0001] This application is a DIVISIONAL application of U.S.
application Ser. No. 09/199,838, filed Nov. 24, 1998, entitled
NOVEL MICROTUBULE-ASSOCIATED PROTEIN, which is a divisional of U.S.
application Ser. No. 08/805,117, filed Feb. 24, 1997, which issued
on Sep. 21, 1999 as U.S. Pat. No. 5,955,312, entitled DNA ENCODING
A NOVEL MICROTUBULE-ASSOCIATED PROTEIN. Both of these applications
are hereby expressly incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a novel human microtubule-associated protein and to
the use of these sequences in the diagnosis, prevention, and
treatment of disorders associated with cell proliferation.
BACKGROUND OF THE INVENTION
[0003] Microtubules are members of an array of fibrous cytoskeleton
proteins which control cell strength and molecular movement within
the cell. In particular, microtubules are critical to chromosomal
movement during cell division. Microtubules are comprised of
tubulin subunits which form a diverse array of both permanent and
transient structures. The processes of microtubule assembly and
disassembly are dynamic and can be affected by various factors
including temperature, anti-cancer drugs such as colchicine and
taxol, and microtubule-associated proteins (MAPs). MAPs are
involved in the formation and the stabilization of
microtubules.
[0004] One major family of MAPs, assembly MAPs, can be identified
in neurons as well as non-neuronal cells. They are responsible for
cross-linking microtubules in the cytosol. These MAPs are organized
into two domains: a basic microtubule-binding domain and an acidic
projection domain. The projection domain is the binding site for
membranes, intermediate filaments, or other microtubules. Based on
sequence analysis, assembly MAPs can be further grouped into two
types: Type I MAPs include MAP1A and MAP1B; and Type II MAPs
include MAP2a, MAP2b, MAP2c, MAP4, and Tau.
[0005] Type I MAPs are large, filamentous molecules that co-purify
with microtubules and are abundantly expressed in brain and testis.
They contain several repeats of the amino acid sequence KKEX. This
positively charged motif binds and neutralizes negatively charged
tubulin, thus stabilizing tubulin by eliminating the repulsion
between tubulin subunits. MAP1A and MAP1B are composed of a heavy
chain and multiple light chains. Two of the light chains, light
chain 1 (LC1) and light chain 2 (LC2), are derived from the 3' end
of the open reading frames that encode the MAP1A and MAP1B heavy
chains.
[0006] A third light chain, LC3, has recently been characterized in
rat and mouse (Mann, S. S. et al. (1994) J. Biol. Chem.
269:11492-11497). LC3 is a 16.4 kDa molecule that binds MAP1A,
MAP1B, and microtubules. It is suggested that LC3 is synthesized
from a source other than the MAP1A or MAP1B transcripts, and the
expression of LC3 may be important in regulating the microtubule
binding activity of MAP1A and MAP1B during cell proliferation.
[0007] The discovery of proteins related to a human
microtubule-associated protein and the oligonucleotides encoding
them satisfies a need in the art by providing new compositions
which are useful in diagnosing, preventing, and treating disorders
associated with cell proliferation.
SUMMARY OF THE INVENTION
[0008] The present invention features a novel human
microtubule-associated protein hereinafter designated hLC3 and
characterized as having similarity to MAP LC3 of Norway rat.
[0009] Accordingly, the invention features a substantially purified
hLC3 having the amino acid sequence shown in SEQ ID NO:1.
[0010] One aspect of the invention features isolated and
substantially purified polynucleotides that encode hLC3. In a
particular aspect, the polynucleotide is the nucleotide sequence of
SEQ ID NO:2.
[0011] The invention also relates to a polynucleotide sequence
comprising the complement of SEQ ID NO:2 or variants thereof. In
addition, the invention features polynucleotide sequences which
hybridize under stringent conditions to SEQ ID NO:2.
[0012] The invention additionally features nucleic acid sequences
encoding polypeptides, oligonucleotides, peptide nucleic acids
(PNA), fragments, portions or antisense molecules thereof, and
expression vectors and host cells comprising polynucleotides that
encode hLC3. The present invention also features antibodies which
bind specifically to hLC3, and pharmaceutical compositions
comprising substantially purified hLC3. The invention also features
methods for treating disorders associated with cell proliferation
using antagonists of hLC3 and for stimulating cell proliferation
using hLC3 or agonists of hLC3.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows the amino acid sequence (SEQ ID NO:1) and
nucleic acid sequence (SEQ ID NO:2) of hLC3. The alignment was
produced using MACDNASIS PRO software (Hitachi Software Engineering
Co., Ltd., San Bruno, Calif.).
[0014] FIG. 2 shows the amino acid sequence alignments between hLC3
(SEQ ID NO:1) and rat MAP LC3 (GI 455109; SEQ ID NO:3). The
alignment was produced using the multi sequence alignment program
of DNASTAR software (DNASTAR Inc, Madison Wis.).
[0015] FIGS. 3A and 3B show the hydrophobicity plots (MACDNASIS PRO
software) for hLC3 (SEQ ID NO: 1) and rat MAP LC3 (GI 455109; SEQ
ID NO:3), respectively; the positive X axis reflects amino acid
position, and the negative Y axis, hydrophobicity.
DESCRIPTION OF THE INVENTION
[0016] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described as these may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0017] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to the "antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the cell lines, vectors, and methodologies which are reported in
the publications which might be used in connection with the
invention. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
[0019] Definitions
[0020] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single- or double-stranded, and represent the sense or
antisense strand. Similarly, "amino acid sequence" as used herein
refers to an oligopeptide, peptide, polypeptide, or protein
sequence, and fragments or portions thereof, and to naturally
occurring or synthetic molecules.
[0021] Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms, such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0022] "Peptide nucleic acid", as used herein, refers to a molecule
which comprises an oligomer to which an amino acid residue, such as
lysine, and an amino group have been added. These small molecules,
also designated anti-gene agents, stop transcript elongation by
binding to their complementary strand of nucleic acid (Nielsen, P.
E. et al. (1993) Anticancer Drug Des. 8:53-63).
[0023] hLC3, as used herein, refers to the amino acid sequences of
substantially purified hLC3 obtained from any species, particularly
mammalian, including bovine, ovine, porcine, murine, equine, and
preferably human, from any source whether natural, synthetic,
semi-synthetic, or recombinant.
[0024] "Consensus", as used herein, refers to a nucleic acid
sequence which has been resequenced to resolve uncalled bases, or
which has been extended using XL-PCR (Perkin Elmer, Norwalk, Conn.)
in the 5' and/or the 3' direction and resequenced, or which has
been assembled from the overlapping sequences of more than one
Incyte clone using the GEL VIEW Fragment Assembly system (GCG,
Madison, Wis.), or which has been both extended and assembled.
[0025] A "variant" of hLC3, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties, e.g., replacement of
leucine with isoleucine. More rarely, a variant may have
"nonconservative" changes, e.g., replacement of a glycine with a
tryptophan. Similar minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
[0026] A "deletion", as used herein, refers to a change in either
amino acid or nucleotide sequence in which one or more amino acid
or nucleotide residues, respectively, are absent.
[0027] An "insertion" or "addition", as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid or nucleotide residues,
respectively, as compared to the naturally occurring molecule.
[0028] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0029] The term "biologically active", as used herein, refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
hLC3, or any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0030] The term "agonist", as used herein, refers to a molecule
which, when bound to hLC3, causes a change in hLC3 which modulates
the activity of hLC3. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to hLC3.
[0031] The terms "antagonist" or "inhibitor", as used herein, refer
to a molecule which, when bound to hLC3, blocks or modulates the
biological or immunological activity of hLC3. Antagonists and
inhibitors may include proteins, nucleic acids, carbohydrates, or
any other molecules which bind to hLC3.
[0032] The term "modulate", as used herein, refers to a change or
an alteration in the biological activity of hLC3. Modulation may be
an increase or a decrease in protein activity, a change in binding
characteristics, or any other change in the biological, functional
or immunological properties of hLC3.
[0033] The term "mimetic", as used herein, refers to a molecule,
the structure of which is developed from knowledge of the structure
of hLC3 or portions thereof and, as such, is able to effect some or
all of the actions of human microtubule-associated protein-like
molecules.
[0034] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding hLC3 or the
encoded hLC3. Illustrative of such modifications would be
replacement of hydrogen by an alkyl, acyl, or amino group. A
nucleic acid derivative would encode a polypeptide which retains
essential biological characteristics of the natural molecule.
[0035] The term "substantially purified", as used herein, refers to
nucleic or amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and most preferably 90% free from other
components with which they are naturally associated.
[0036] "Amplification" as used herein refers to the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y.).
[0037] The term "hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0038] The term "hybridization complex", as used herein, refers to
a complex formed between two nucleic acid sequences by virtue of
the formation of hydrogen bonds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C.sub.0t or R.sub.0t analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized on a solid support (e.g., membranes, filters,
chips, pins or glass slides to which cells have been fixed for in
situ hybridization).
[0039] The terms "complementary" or "complementarity", as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For
example, for the sequence "A-G-T" binds to the complementary
sequence "T-C-A". Complementarity between two single-stranded
molecules may be "partial", in which only some of the nucleic acids
bind, or it may be complete when total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands.
[0040] The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology
(i.e., identity). A partially complementary sequence is one that at
least partially inhibits an identical sequence from hybridizing to
a target nucleic acid; it is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence or probe to the target sequence
under conditions of low stringency. This is not to say that
conditions of low stringency are such that non-specific binding is
permitted; low stringency conditions require that the binding of
two sequences to one another be a specific (i.e., selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% identity); in
the absence of non-specific binding, the probe will not hybridize
to the second non-complementary target sequence.
[0041] As known in the art, numerous equivalent conditions may be
employed to comprise either low or high stringency conditions.
Factors such as the length and nature (DNA, RNA, base composition)
of the sequence, nature of the target (DNA, RNA, base composition,
presence in solution or immobilization, etc.), and the
concentration of the salts and other components (e.g., the presence
or absence of formamide, dextran sulfate and/or polyethylene
glycol) are considered and the hybridization solution may be varied
to generate conditions of either low or high stringency different
from, but equivalent to, the above listed conditions.
[0042] The term "stringent conditions", as used herein, is the
"stringency" which occurs within a range from about Tm-5.degree. C.
(5.degree. C. below the melting temperature (Tm) of the probe) to
about 20.degree. C. to 25.degree. C. below Tm. As will be
understood by those of skill in the art, the stringency of
hybridization may be altered in order to identify or detect
identical or related polynucleotide sequences.
[0043] The term "antisense", as used herein, refers to nucleotide
sequences which are complementary to a specific DNA or RNA
sequence. The term "antisense strand" is used in reference to a
nucleic acid strand that is complementary to the "sense" strand.
Antisense molecules may be produced by any method, including
synthesis by ligating the gene(s) of interest in a reverse
orientation to a viral promoter which permits the synthesis of a
complementary strand. Once introduced into a cell, this transcribed
strand combines with natural sequences produced by the cell to form
duplexes. These duplexes then block either the further
transcription or translation. In this manner, mutant phenotypes may
be generated. The designation "negative" is sometimes used in
reference to the antisense strand, and "positive" is sometimes used
in reference to the sense strand.
[0044] The term "portion", as used herein, with regard to a protein
(as in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from four amino acid
residues to the entire amino acid sequence minus one amino acid.
Thus, a protein "comprising at least a portion of the amino acid
sequence of SEQ ID NO:1" encompasses the full-length human hLC3 and
fragments thereof.
[0045] "Transformation", as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the host cell being transformed and may include, but is not
limited to, viral infection, electroporation, lipofection, and
particle bombardment. Such "transformed" cells include stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome. They also include cells which
transiently express the inserted DNA or RNA for limited periods of
time.
[0046] The term "antigenic determinant", as used herein, refers to
that portion of a molecule that makes contact with a particular
antibody (i.e., an epitope). When a protein or fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to a given region or three-dimensional structure on
the protein; these regions or structures are referred to as
antigenic determinants. An antigenic determinant may compete with
the intact antigen (i.e., the immunogen used to elicit the immune
response) for binding to an antibody.
[0047] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody and a
protein or peptide, mean that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words, the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. For example, if an antibody is specific for
epitope "A", the presence of a protein containing epitope A (or
free, unlabeled A) in a reaction containing labeled "A" and the
antibody will reduce the amount of labeled A bound to the
antibody.
[0048] The term "sample", as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acid
encoding hLC3 or fragments thereof may comprise a cell, chromosomes
isolated from a cell (e.g., a spread of metaphase chromosomes),
genomic DNA (in solution or bound to a solid support such as for
Southern analysis), RNA (in solution or bound to a solid support
such as for northern analysis), cDNA (in solution or bound to a
solid support), an extract from cells or a tissue, and the
like.
[0049] The term "correlates with expression of a polynucleotide",
as used herein, indicates that the detection of the presence of
ribonucleic acid that is similar to SEQ ID NO:2 by northern
analysis is indicative of the presence of mRNA encoding hLC3 in a
sample and thereby correlates with expression of the transcript
from the polynucleotide encoding the protein.
[0050] "Alterations" in the polynucleotide of SEQ ID NO: 2, as used
herein, comprise any alteration in the sequence of polynucleotides
encoding hLC3 including deletions, insertions, and point mutations
that may be detected using hybridization assays. Included within
this definition is the detection of alterations to the genomic DNA
sequence which encodes hLC3 (e.g., by alterations in the pattern of
restriction fragment length polymorphisms capable of hybridizing to
SEQ ID NO:2), the inability of a selected fragment of SEQ ID NO:2
to hybridize to a sample of genomic DNA (e.g., using
allele-specific oligonucleotide probes), and improper or unexpected
hybridization, such as hybridization to a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
hLC3 (e.g., using fluorescent in situ hybridization [FISH] to
metaphase chromosomes spreads).
[0051] As used herein, the term "antibody" refers to intact
molecules as well as fragments thereof, such as Fa, F(ab').sub.2,
and Fv, which are capable of binding the epitopic determinant
Antibodies that bind hLC3 polypeptides can be prepared using intact
polypeptides or fragments containing small peptides of interest as
the immunizing antigen. The polypeptide or peptide used to immunize
an animal can be derived from the transition of RNA or synthesized
chemically, and can be conjugated to a carrier protein, if desired.
Commonly used carriers that are chemically coupled to peptides
include bovine serum albumin and thyroglobulin. The coupled peptide
is then used to immunize the animal (e.g., a mouse, a rat, or a
rabbit).
[0052] The term "humanized antibody", as used herein, refers to
antibody molecules in which amino acids have been replaced in the
non-antigen binding regions in order to more closely resemble a
human antibody, while still retaining the original binding
ability.
[0053] The Invention
[0054] The invention is based on the discovery of a novel human
microtubule-associated protein (hLC3), the polynucleotides encoding
hLC3, and the use of these compositions for the diagnosis,
prevention, or treatment of disorders associated with cell
proliferation.
[0055] Nucleic acids encoding the human hLC3 of the present
invention were first identified in Incyte Clone 1441378 from a
thyroid cDNA library (THYRNOT03) through a computer-generated
search for amino acid sequence alignments. A consensus sequence,
SEQ ID NO:2, was derived from the following overlapping and/or
extended nucleic acid sequences 3: Incyte Clones 855614
(NGANNOT01), 927600 (BRAINOT04), 1441378 (THYRNOT03), and 1962012
(BRSTNOT04).
[0056] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in FIG.
1. hLC3 is 121 amino acid residues in length. Motif search reveals
a number of potential chemical modification sites: an
N-glycosylation site at N59; a protein kinase C phosphorylation
site at S3; a casein kinase II phosphorylation site at S12; and a
tyrosine kinase phosphorylation site at Y110. hLC3 has 82% chemical
and structural identity with rat LC3 (GI 455109; SEQ ID NO:3), and
they also have similar hydrophobicity plots (FIGS. 3A and 3B).
Northern analysis shows the expression of hLC3 in various
libraries. Of particular note is the expression of hLC3 in a
significant number of cancerous cells or tissues.
[0057] The invention also encompasses hLC3 variants. A preferred
hLC3 variant is one having at least 80%, and more preferably 90%,
amino acid sequence identity to the hLC3 amino acid sequence (SEQ
ID NO:1). A most preferred hLC3 variant is one having at least 95%
amino acid sequence identity to SEQ ID NO:1.
[0058] The invention also encompasses polynucleotides which encode
hLC3. Accordingly, any nucleic acid sequence which encodes the
amino acid sequence of hLC3 can be used to generate recombinant
molecules which express hLC3. In a particular embodiment, the
invention encompasses the polynucleotide comprising the nucleic
acid sequence of SEQ ID) NO:2 as shown in FIG. 1.
[0059] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
nucleotide sequences encoding hLC3, some bearing minimal homology
to the nucleotide sequences of any known and naturally occurring
gene, may be produced. Thus, the invention contemplates each and
every possible variation of nucleotide sequence that could be made
by selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring hLC3, and all such variations are to be considered as
being specifically disclosed.
[0060] Although nucleotide sequences which encode hLC3 and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring hLC3 under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding hLC3 or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding hLC3 and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0061] The invention also encompasses production of DNA sequences,
or portions thereof, which encode hLC3 and its derivatives,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art at the time of the filing of this application. Moreover,
synthetic chemistry may be used to introduce mutations into a
sequence encoding hLC3 or any portion thereof.
[0062] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in SEQ ID NO:2, under
various conditions of stringency. Hybridization conditions are
based on the melting temperature (Tm) of the nucleic acid binding
complex or probe, as taught in Wahl, G. M. and S. L. Berger (1987;
Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods
Enzymol. 152:507-511), and may be used at a defined stringency.
[0063] Altered nucleic acid sequences encoding hLC3 which are
encompassed by the invention include deletions, insertions, or
substitutions of different nucleotides resulting in a
polynucleotide that encodes the same or a functionally equivalent
hLC3. The encoded protein may also contain deletions, insertions,
or substitutions of amino acid residues which produce a silent
change and result in a functionally equivalent hLC3. Deliberate
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
biological activity of hLC3 is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid;
positively charged amino acids may include lysine and arginine; and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; phenylalanine and tyrosine.
[0064] Also included within the scope of the present invention are
alleles of the genes encoding hLC3. As used herein, an "allele" or
"allelic sequence" is an alternative form of the gene which may
result from at least one mutation in the nucleic acid sequence.
Alleles may result in altered mRNAs or polypeptides whose structure
or function may or may not be altered. Any given gene may have
none, one, or many allelic forms. Common mutational changes which
give rise to alleles are generally ascribed to natural deletions,
additions, or substitutions of nucleotides. Each of these types of
changes may occur alone, or in combination with the others, one or
more times in a given sequence.
[0065] Methods for DNA sequencing which are well known and
generally available in the art may be used to practice any
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, SEQUENASE (US
Biochemical Corp, Cleveland, Ohio.), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of recombinant polymerases and proofreading
exonucleases such as the ELONGASE Amplification System marketed by
Gibco BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,
Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,
Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).
[0066] The nucleic acid sequences encoding hLC3 may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences such as
promoters and regulatory elements. For example, one method which
may be employed, "restriction-site" PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA
is first amplified in the presence of primer to linker sequence and
a primer specific to the known region. The amplified sequences are
then subjected to a second round of PCR with the same linker primer
and another specific primer internal to the first one. Products of
each round of PCR are transcribed with an appropriate RNA
polymerase and sequenced using reverse transcriptase.
[0067] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region (Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186). The primers may be designed
using OLIGO 4.06 Primer Analysis software (National Biosciences
Inc., Plymouth, Minn.), or another appropriate program, to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to
anneal to the target sequence at temperatures about
68.degree.-72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0068] Another method which may be used is capture PCR which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119). In this method,
multiple restriction enzyme digestions and ligations may also be
used to place an engineered double-stranded sequence into an
unknown portion of the DNA molecule before performing PCR.
[0069] Another method which may be used to retrieve unknown
sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries to walk in genomic DNA (Clontech, Palo
Alto, Calif.). This process avoids the need to screen libraries and
is useful in finding intron/exon junctions. When screening for
full-length cDNAs, it is preferable to use libraries that have been
size-selected to include larger cDNAs. Also, random-primed
libraries are preferable, in that they will contain more sequences
which contain the 5' regions of genes. Use of a randomly primed
library may be especially preferable for situations in which an
oligo d(T) library does not yield a full-length cDNA. Genomic
libraries may be useful for extension of sequence into the 5' and
3' non-transcribed regulatory regions.
[0070] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and detection of the emitted
wavelengths by a charge coupled device camera. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer) and
the entire process from loading of samples to computer analysis and
electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0071] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode hLC3, or fusion
proteins or functional equivalents thereof, may be used in
recombinant DNA molecules to direct expression of hLC3 in
appropriate host cells. Due to the inherent degeneracy of the
genetic code, other DNA sequences which encode substantially the
same or a functionally equivalent amino acid sequence may be
produced and these sequences may be used to clone and express
hLC3.
[0072] As will be understood by those of skill in the art, it may
be advantageous to produce hLC3-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce a
recombinant RNA transcript having desirable properties, such as a
half-life which is longer than that of a transcript generated from
the naturally occurring sequence.
[0073] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter hLC3 encoding sequences for a variety of reasons, including
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, or introduce mutations,
and so forth.
[0074] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding hLC3 may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of hLC3 activity, it may
be useful to encode a chimeric hLC3 protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the hLC3
encoding sequence and the heterologous protein sequence, so that
hLC3 may be cleaved and purified away from the heterologous
moiety.
[0075] In another embodiment, sequences encoding hLC3 may be
synthesized, in whole or in part, using chemical methods well known
in the art (see Caruthers, M. H. et al. (1980) Nucleic Acids Symp.
Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232). Alternatively, the protein itself may be produced using
chemical methods to synthesize the amino acid sequence of hLC3, or
a portion thereof. For example, peptide synthesis can be performed
using various solid-phase techniques (Roberge, J. Y. et al. (1995)
Science 269:202-204) and automated synthesis may be achieved, for
example, using the ABI 431A Peptide Synthesizer (Perkin Elmer).
[0076] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
WH Freeman and Co., New York, N.Y.). The composition of the
synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). Additionally, the amino acid sequence of hLC3, or any part
thereof, may be altered during direct synthesis and/or combined
using chemical methods with sequences from other proteins, or any
part thereof, to produce a variant polypeptide.
[0077] In order to express a biologically active hLC3, the
nucleotide sequences encoding hLC3 or functional equivalents, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0078] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding hLC3 and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.
[0079] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding hLC3. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems.
[0080] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1
plasmid (Gibco BRL) and the like may be used. The baculovirus
polyhedrin promoter may be used in insect cells. Promoters or
enhancers derived from the genomes of plant cells (e.g., heat
shock, RUBISCO; and storage protein genes) or from plant viruses
(e.g., viral promoters or leader sequences) may be cloned into the
vector. In mammalian cell systems, promoters from mammalian genes
or from mammalian viruses are preferable. If it is necessary to
generate a cell line that contains multiple copies of the sequence
encoding hLC3, vectors based on SV40 or EBV may be used with an
appropriate selectable marker.
[0081] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for hLC3 R. For example,
when large quantities of hLC3 are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, the multifunctional E. coli
cloning and expression vectors such as BLUESCRIPT (Stratagene), in
which the sequence encoding hLC3 may be ligated into the vector in
frame with sequences for the amino-terminal Met and the subsequent
7 residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509); and the like. pGEX vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems may be designed to include heparin, thrombin, or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0082] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Bitter, G. A. et al. (1987) Methods
Enzymol. 153:516-544.
[0083] In cases where plant expression vectors are used, the
expression of sequences encoding hLC3 may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196).
[0084] An insect system may also be used to express hLC3. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding hLC3 may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of hLC3 will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses may then be used to
infect, for example, S. frugiperda cells or Trichoplusia larvae in
which hLC3 may be expressed (Engelhard, E. K. et al. (1994) Proc.
Natl. Acad. Sci. USA 91:3224-3227).
[0085] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding hLC3 may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing hLC3 in
infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl.
Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0086] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding hLC3. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding hLC3, its initiation codon, and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a portion
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0087] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have
specific cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
[0088] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express hLC3 may be transformed using expression
vectors which may contain viral origins of replication and/or
endogenous expression elements and a selectable marker gene on the
same or on a separate vector. Following the introduction of the
vector, cells may be allowed to grow for 1-2 days in an enriched
media before they are switched to selective media. The purpose of
the selectable marker is to confer resistance to selection, and its
presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be proliferated using tissue culture
techniques appropriate to the cell type.
[0089] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-232) and adenine phosphoribosyl-transferase (Lowy, I.
et al. (1980) Cell 22:817-823) genes which can be employed in
tk.sup.- and apr.sup.- cells, respectively. Also, antimetabolite,
antibiotic or herbicide resistance can be used as the basis for
selection; for example, dhfr which confers resistance to
methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA
77:3567-3570); npt, which confers resistance to the aminoglycosides
neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.
150:1-14) and als and pat, which confer resistance to chlorsulfuron
and phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
USA 85:8047-8051). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta. glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0090] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequence encoding hLC3 is inserted within a marker gene sequence,
recombinant cells containing sequences encoding hLC3 can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding hLC3
under the control of a single promoter. Expression of the marker
gene in response to induction or selection usually indicates
expression of the tandem gene as well.
[0091] Alternatively, host cells which contain the nucleic acid
sequence encoding hLC3 and express hLC3 may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein.
[0092] The presence of polynucleotide sequences encoding hLC3 can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or portions or fragments of polynucleotides encoding
hLC3. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding hLC3
to detect transformants containing DNA or RNA encoding hLC3. As
used herein "oligonucleotides" or "oligomers" refer to a nucleic
acid sequence of at least about 10 nucleotides and as many as about
60 nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides, which can be used as a probe or
amplimer.
[0093] A variety of protocols for detecting and measuring the
expression of hLC3, using either polyclonal or monoclonal
antibodies specific for the protein are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on hLC3 is preferred, but
a competitive binding assay may be employed. These and other assays
are described, among other places, in Hampton, R. et al. (1990;
Serological Methods, a Laboratory Manual, APS Press, St. Paul,
Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216).
[0094] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding hLC3 include oligolabeling, nick
translation, end-labeling or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding hLC3 R, or any
portions thereof may be cloned into a vector for the production of
an mRNA probe. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a
variety of commercially available kits (Pharmacia & Upjohn,
(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical
Corp., Cleveland, Ohio)). Suitable reporter molecules or labels,
which may be used, include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0095] Host cells transformed with nucleotide sequences encoding
hLC3 may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a recombinant cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode hLC3 may be designed to
contain signal sequences which direct secretion of hLC3 through a
prokaryotic or eukaryotic cell membrane. Other recombinant
constructions may be used to join sequences encoding hLC3 to
nucleotide sequence encoding a polypeptide domain which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). The inclusion of cleavable linker sequences such
as those specific for Factor XA or enterokinase (Invitrogen, San
Diego, Calif.) between the purification domain and hLC3 may be used
to facilitate purification. One such expression vector provides for
expression of a fusion protein containing hLC3 and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography) as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3:263-281) while the enterokinase cleavage site provides a
means for purifying hLC3 from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0096] In addition to recombinant production, fragments of hLC3 may
be produced by direct peptide synthesis using solid-phase
techniques (Merrifield, J. (1963) J. Am. Chem. Soc. 85:2149-2154).
Protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various
fragments of hLC3 may be chemically synthesized separately and
combined using chemical methods to produce the full length
molecule.
[0097] Therapeutics
[0098] hLC3 shares 82% chemical and structural identity with rat
MAP LC3. Northern analysis shows that the expression of hLC3 is
associated with disorders of cell proliferation, and in particular,
cancer.
[0099] Therefore, in one embodiment, hLC3 or a fragment or
derivative thereof may be added to a cell to stimulate cell
proliferation. In another embodiment, a vector capable of
expressing hLC3, or a fragment or a derivative thereof, may also be
transformed into a cell to stimulate cell proliferation. In another
embodiment, agonists which are specific for hLC3 may be used to
stimulate or prolong the activity of hLC3 in stimulating cell
proliferation. Tissues or organs generated by these methods may be
introduced or transplanted into a subject for the purpose of
treating a variety of diseases.
[0100] In another embodiment, antagonists or inhibitors of hLC3 may
be administered to a subject to treat or prevent disorders
associated with cell proliferation. Such disorders include various
types of cancer including, but not limited to, that of the bladder,
bone, brain, breast, colon, heart, kidney, liver, lung, ovary,
pancreas, paraganglia, parathyroid, prostate, skin, small
intestine, testis, thyroid, tongue, and uterus. Antibodies which
are specific for hLC3 may be used directly as an antagonist, or
indirectly as a targeting or delivery mechanism for bringing a
pharmaceutical agent to cells or tissue which express hLC3.
[0101] In another embodiment, a vector expressing antisense of the
polynucleotide encoding hLC3 may be administered to a subject to
treat or prevent disorders associated with cell proliferation
including, but not limited to, those listed above.
[0102] In other embodiments, any of the therapeutic proteins,
antagonists, antibodies, agonists, antisense sequences or vectors
described above may be administered in combination with other
appropriate therapeutic agents. Selection of the appropriate agents
for use in combination therapy may be made by one of ordinary skill
in the art, according to conventional pharmaceutical principles.
The combination of therapeutic agents may act synergistically to
effect the treatment or prevention of the various disorders
described above. Using this approach, one may be able to achieve
therapeutic efficacy with lower dosages of each agent, thus
reducing the potential for adverse side effects.
[0103] Antagonists or inhibitors of hLC3 may be produced using
methods which are generally known in the art. In particular,
purified hLC3 may be used to produce antibodies or to screen
libraries of pharmaceutical agents to identify those which
specifically bind hLC3.
[0104] The antibodies to hLC3 may be generated using methods that
are well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, single chain, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies, (i.e., those which inhibit dimer
formation) are especially preferred for therapeutic use.
[0105] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with hLC3 or any fragment or oligopeptide thereof which
has immunogenic properties. Depending on the host species, various
adjuvants may be used to increase immunological response. Such
adjuvants include, but are not limited to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable.
[0106] It is preferred that the peptides, fragments, or
oligopeptides used to induce antibodies to hLC3 have an amino acid
sequence consisting of at least five amino acids, and more
preferably at least 10 amino acids. It is also preferable that they
are identical to a portion of the amino acid sequence of the
natural protein, and they may contain the entire amino acid
sequence of a small, naturally occurring molecule. Short stretches
of hLC3 amino acids may be fused with those of another protein such
as keyhole limpet hemocyanin and antibody produced against the
chimeric molecule.
[0107] Monoclonal antibodies to hLC3 may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120).
[0108] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.
S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985)
Nature 314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce hLC3-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic
composition, may be generated by chain shuffling from random
combinatorial immunoglobin libraries (Burton, D. R. (1991) Proc.
Natl. Acad. Sci. USA 88:11120-11123).
[0109] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991)
Nature 349:293-299).
[0110] Antibody fragments which contain specific binding sites for
hLC3 may also be generated. For example, such fragments include,
but are not limited to, the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 254:1275-1281).
[0111] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between hLC3 and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering hLC3 epitopes
is preferred, but a competitive binding assay may also be employed
(Maddox, supra).
[0112] In another embodiment of the invention, the polynucleotides
encoding hLC3, or any fragment thereof, or antisense molecules, may
be used for therapeutic purposes. In one aspect, antisense to the
polynucleotide encoding hLC3 may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding hLC3. Thus, antisense molecules may be
used to modulate hLC3 activity, or to achieve regulation of gene
function. Such technology is now well known in the art, and sense
or antisense oligomers or larger fragments, can be designed from
various locations along the coding or control regions of sequences
encoding hLC3.
[0113] Expression vectors derived from retro viruses, adenovirus,
herpes or vaccinia viruses, or from various bacterial plasmids may
be used for delivery of nucleotide sequences to the targeted organ,
tissue or cell population. Methods which are well known to those
skilled in the art can be used to construct recombinant vectors
which will express antisense molecules complementary to the
polynucleotides of the gene encoding hLC3. These techniques are
described both in Sambrook et al. (supra) and in Ausubel et al.
(supra).
[0114] Genes encoding hLC3 can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide or fragment thereof which encodes hLC3. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector and even longer if appropriate replication elements are part
of the vector system.
[0115] As mentioned above, modifications of gene expression can be
obtained by designing antisense molecules, DNA, RNA, or PNA, to the
control regions of the gene encoding hLC3, i.e., the promoters,
enhancers, and introns. Oligonucleotides derived from the
transcription initiation site, e.g., between positions -10 and +10
from the start site, are preferred. Similarly, inhibition can be
achieved using "triple helix" base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I.
Carr, Molecular and Immunologic Approaches, Futura Publishing Co.,
Mt. Kisco, N.Y.). The antisense molecules may also be designed to
block translation of mRNA by preventing the transcript from binding
to ribosomes.
[0116] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples which may be used include engineered hammerhead
motif ribozyme molecules that can specifically and efficiently
catalyze endonucleolytic cleavage of sequences encoding hLC3.
[0117] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0118] Antisense molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of
nucleic acid molecules. These include techniques for chemically
synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis. Alternatively, RNA molecules may be generated
by in vitro and in vivo transcription of DNA sequences encoding
hLC3. Such DNA sequences may be incorporated into a wide variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA constructs that synthesize antisense RNA
constitutively or inducibly can be introduced into cell lines,
cells, or tissues.
[0119] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0120] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection
and by liposome injections may be achieved using methods which are
well known in the art.
[0121] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0122] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of hLC3, antibodies to hLC3, mimetics, agonists,
antagonists, or inhibitors of hLC3. The compositions may be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0123] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0124] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0125] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0126] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0127] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0128] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0129] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0130] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0131] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0132] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the following: 1-50 mM histidine, 0.1% 2%
sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is
combined with buffer prior to use.
[0133] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of hLC3, such
labeling would include amount, frequency, and method of
administration.
[0134] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0135] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0136] A therapeutically effective dose refers to that amount of
active ingredient, for example hLC3 or fragments thereof,
antibodies of hLC3, agonists, antagonists or inhibitors of hLC3,
which ameliorates the symptoms or condition. Therapeutic efficacy
and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50
(the dose therapeutically effective in 50% of the population) and
LD50 (the dose lethal to 50% of the population). The dose ratio
between therapeutic and toxic effects is the therapeutic index, and
it can be expressed as the ratio, LD50/ED50. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies is
used in formulating a range of dosage for human use. The dosage
contained in such compositions is preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0137] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active moiety or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0138] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0139] Diagnostics
[0140] In another embodiment, antibodies which specifically bind
hLC3 may be used for the diagnosis of conditions or diseases
characterized by expression of hLC3, or in assays to monitor
patients being treated with hLC3, agonists, antagonists or
inhibitors. The antibodies useful for diagnostic purposes may be
prepared in the same manner as those described above for
therapeutics. Diagnostic assays for hLC3 include methods which
utilize the antibody and a label to detect hLC3 in human body
fluids or extracts of cells or tissues. The antibodies may be used
with or without modification, and may be labeled by joining them,
either covalently or non-covalently, with a reporter molecule. A
wide variety of reporter molecules which are known in the art may
be used, several of which are described above.
[0141] A variety of protocols including ELISA, RIA, and FACS for
measuring hLC3 are known in the art and provide a basis for
diagnosing altered or abnormal levels of hLC3 expression. Normal or
standard values for hLC3 expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to hLC3 under conditions suitable
for complex formation. The amount of standard complex formation may
be quantified by various methods, but preferably by photometric
means. Quantities of hLC3 expressed in subject, control, and
disease, samples from biopsied tissues are compared with the
standard values. Deviation between standard and subject values
establishes the parameters for diagnosing disease.
[0142] In another embodiment of the invention, the polynucleotides
encoding hLC3 may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, antisense RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of hLC3 may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
hLC3, and to monitor regulation of hLC3 levels during therapeutic
intervention.
[0143] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding hLC3 or closely related molecules, may be used
to identify nucleic acid sequences which encode an hLC3. The
specificity of the probe, whether it is made from a highly specific
region, e.g., 10 unique nucleotides in the 5' regulatory region, or
a less specific region, e.g., especially in the 3' coding region,
and the stringency of the hybridization or amplification (maximal,
high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding hLC3,
alleles, or related sequences.
[0144] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the hLC3 encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
derived from the nucleotide sequence of SEQ ID NO:2 or from genomic
sequence including promoter, enhancer elements, and introns of the
naturally occurring hLC3.
[0145] Means for producing specific hybridization probes for DNAs
encoding hLC3 include the cloning of nucleic acid sequences
encoding hLC3 or hLC3 derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, radionuclides
such as 32P or 35S, or enzymatic labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
[0146] Polynucleotide sequences encoding hLC3 may be used for the
diagnosis of conditions or diseases which are associated with the
expression of hLC3. Examples of such conditions or diseases include
cancer of the bladder, bone, brain, breast, colon, heart, immune
system, kidney, liver, lung, ovary, pancreas, paraganglia,
parathyroid, prostate, skin, small intestine, testis, thyroid,
tongue, and uterus. The polynucleotide sequences encoding hLC3 may
be used in Southern or northern analysis, dot blot, or other
membrane-based technologies; in PCR technologies; or in dip stick,
pin, ELISA or chip assays utilizing fluids or tissues from patient
biopsies to detect altered hLC3 expression. Such qualitative or
quantitative methods are well known in the art.
[0147] In a particular aspect, the nucleotide sequences encoding
hLC3 may be useful in assays that detect activation or induction of
various cancers, particularly those mentioned above. The nucleotide
sequences encoding hLC3 may be labeled by standard methods, and
added to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the biopsied or extracted sample is significantly altered
from that of a comparable control sample, the nucleotide sequences
have hybridized with nucleotide sequences in the sample, and the
presence of altered levels of nucleotide sequences encoding hLC3 in
the sample indicates the presence of the associated disease. Such
assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in animal studies, in clinical
trials, or in monitoring the treatment of an individual
patient.
[0148] In order to provide a basis for the diagnosis of disease
associated with expression of hLC3, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
which encodes hLC3, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease.
[0149] Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0150] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0151] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding hLC3 may involve the use of PCR. Such
oligomers may be chemically synthesized, generated enzymatically,
or produced from a recombinant source. Oligomers will preferably
consist of two nucleotide sequences, one with sense orientation
(5'->3') and another with antisense (3'<-5'), employed under
optimized conditions for identification of a specific gene or
condition. The same two oligomers, nested sets of oligomers, or
even a degenerate pool of oligomers may be employed under less
stringent conditions for detection and/or quantitation of closely
related DNA or RNA sequences.
[0152] Methods which may also be used to quantitate the expression
of hLC3 include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and standard curves onto
which the experimental results are interpolated (Melby, P. C. et
al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al.
(1993) Anal. Biochem. 212:229-236). The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or calorimetric response gives
rapid quantitation.
[0153] In another embodiment of the invention, the nucleic acid
sequences which encode hLC3 may also be used to generate
hybridization probes which are useful for mapping the naturally
occurring genomic sequence. The sequences may be mapped to a
particular chromosome or to a specific region of the chromosome
using well known techniques. Such techniques include FISH, FACS, or
artificial chromosome constructions, such as yeast artificial
chromosomes, bacterial artificial chromosomes, bacterial P1
constructions or single chromosome cDNA libraries as reviewed in
Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J. (1991)
Trends Genet. 7:149-154.
[0154] FISH (as described in Verma et al. (1988) Human Chromosomes:
A Manual of Basic Techniques, Pergamon Press, New York, N.Y.) may
be correlated with other physical chromosome mapping techniques and
genetic map data. Examples of genetic map data can be found in the
1994 Genome Issue of Science (265:1981 f). Correlation between the
location of the gene encoding hLC3 on a physical chromosomal map
and a specific disease, or predisposition to a specific disease,
may help delimit the region of DNA associated with that genetic
disease. The nucleotide sequences of the subject invention may be
used to detect differences in gene sequences between normal,
carrier, or affected individuals.
[0155] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms, or parts
thereof, by physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, for example, AT to 11q22-23 (Gatti, R. A. et al.
(1988) Nature 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
The nucleotide sequence of the subject invention may also be used
to detect differences in the chromosomal location due to
translocation, inversion, etc. among normal, carrier, or affected
individuals.
[0156] In another embodiment of the invention, hLC3, its catalytic
or immunogenic fragments or oligopeptides thereof, can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes, between hLC3 and the agent being tested, may be
measured.
[0157] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, as applied to
hLC3 large numbers of different small test compounds are
synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with hLC3, or
fragments thereof, and washed. Bound hLC3 is then detected by
methods well known in the art. Purified hLC3 can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0158] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding hLC3 specifically compete with a test compound for binding
hLC3. In this manner, the antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with hLC3.
[0159] In additional embodiments, the nucleotide sequences which
encode hLC3 may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0160] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0161] I. THYRNOT03 cDNA Library Construction
[0162] The THYRNOT03 cDNA library was constructed from
microscopically normal thyroid adenoma tissue obtained from a
28-year-old Caucasian female diagnosed with nontoxic uninodular
goiter, toxic multinodular goiter, and dysphagia. The patient
history included alcohol use, non-obstructed galactorrhea, profound
anemia, pure hypercholesterolemia, and
tonsillectomy/adenoidectomy.
[0163] The frozen tissue was homogenized and lysed using a
Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments,
Westbury, N.J.) in guanidinium isothiocyanate solution. The lysate
was centrifuged over a 5.7 M CsCl cushion using a Beckman SW28
rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for
18 hours at 25,000 rpm at ambient temperature. The RNA was
extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium
acetate and 2.5 volumes of ethanol, resuspended in RNAse-free
water, and DNase treated at 37.degree. C. Extraction and
precipitation were repeated as before. The mRNA was isolated with
the QIAGEN OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.) and used
to construct the cDNA library.
[0164] The mRNA was handled according to the recommended protocols
in the SuperScript Plasmid System for cDNA Synthesis and Plasmid
Cloning (Cat. #18248-013, Gibco/BRL, Gaithersburg, Md.). cDNAs were
fractionated on a Sepharose CL4B column (Cat. #275105-01,
Pharmacia), and those cDNAs exceeding 400 bp were ligated into
pINCY. The plasmid pINCY was subsequently transformed into DH5a
competent cells (Cat. #18258-012, Gibco/BRL).
[0165] II. Isolation and Sequencing of cDNA Clones
[0166] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 Plasmid Kit (Catalog #26173, QIAGEN, Inc.). This
kit enabled the simultaneous purification of 96 samples in a
96-well block using multi-channel reagent dispensers. The
recommended protocol was employed except for the following changes:
1) the bacteria were cultured in 1 ml of sterile Terrific Broth
(Catalog #22711, LIFE TECHNOLOGIES, Gaithersburg, Md.) with
carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after
inoculation, the cultures were incubated for 19 hours and at the
end of incubation, the cells were lysed with 0.3 ml of lysis
buffer; and 3) following isopropanol precipitation, the plasmid DNA
pellet was resuspended in 0.1 ml of distilled water. After the last
step in the protocol, samples were transferred to a 96-well block
for storage at 4.degree. C.
[0167] The cDNAs were sequenced by the method of Sanger et al.
(1975, J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers
(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems
377 DNA Sequencing Systems; and the reading frame was
determined.
[0168] Most of the sequences disclosed herein were sequenced
according to standard ABI protocols, using ABI kits (Cat. Nos.
79345, 79339, 79340, 79357, 79355). The solution volumes were used
at 0.25.times.-10.times. concentrations. Some of the sequences
disclosed herein were sequenced using different solutions and dyes
which, unless otherwise noted, came from Amersham Life Science
(Cleveland, Ohio.).
[0169] First, stock solutions were prepared with HPLC water. The
following solutions were each mixed by vortexing for 2 min: 1)
Tris-EDTA (TE) Buffer was prepared by adding 49 ml water to 1 ml
50.times. Tris-EDTA concentrate, and 2) 10% Reaction Buffer was
prepared by adding 45 ml water to 5 ml Concentrated Thermo
Sequenase (TS) Reaction Buffer.
[0170] Second, 0.2 .mu.M energy transfer (ET) primers were prepared
in the following manner. Each primer tube was centrifuged prior to
opening to assure that all primer powder was on the bottom of the
tube. After each solubilization step, the mixture was vortexed for
2 min and then centrifuged for about 10 sec in a table-top
centrifuge. 1 ml of 1.times. TE was added to each primer powder;
adenine and cytosine dissolved primers (5-carboxyrhodamine-6G (R6G)
and 6-carboxyfluorescein (FAM), respectively), were diluted with 9
ml 1.times. TE. Guanine and thymine dyes
(N,N,N',N"-tetramethyl-6-carboxyrhodamine (TAM) and
6-carboxy-X-rhodamine (ROX), respectively) were diluted with 19 ml
1.times. TE.
[0171] Next, the sequencing reaction ready mix was prepared as
follows: 1) nucleotides A and C (8 ml of each) were added to 6 ml
ET primer and 18 ml TS reaction buffer; and 2) nucleotides G and T
(8 ml of each) were added to 6 ml ET primer and 18 ml TS reaction
buffer.
[0172] After vortexing for 2 min and centrifuging for 20 sec, the
resulting solution was divided into tubes in volumes of 8 ml per
tube in order to make 1.times. (A,C) and 2.times. (G,T)
solutions.
[0173] Prior to thermal cycling, each nucleotide was individually
mixed with DNA template in the following proportions:
1 Reagent A (.mu.L) C (.mu.L) G (.mu.L) T (.mu.L) Reaction ready
premix 2 2 4 4 DNA template 1 1 2 2 Total Volume 3 3 6 6
[0174] These solutions undergo the usual thermal cycling:
[0175] 1. Rapid thermal ramp to 94.degree. C. (94.degree. C. for 20
sec)*
[0176] 2. Rapid thermal ramp to 50.degree. C. (50.degree. C. for 40
sec)*
[0177] 3. Rapid thermal ramp to 68.degree. C. (68.degree. C. for 60
sec)*
[0178] * Steps 1, 2, and 3 were repeated for 15 cycles
[0179] 4. Rapid thermal ramp to 94.degree. C. (94.degree. C. for 20
sec)**
[0180] 5. Rapid thermal ramp to 68.degree. C. (68.degree. C. for 60
sec)**
[0181] ** Steps 4 and 5 were repeated for 15 cycles
[0182] 6. Rapid thermal ramp to 4.degree. C. and hold until ready
to combine.
[0183] After thermal cycling, the A, C, G, and T reactions with
each DNA template were combined. Then, 50 .mu.L 100% ethanol was
added and the solution was spun at 4.degree. C. for 30 min. The
supernatant was decanted and the pellet was rinsed with 100 .mu.L
70% ethanol. After being spun for 15 min the supernatant was
discarded and the pellet was dried for 15 min under vacuum. The DNA
sample was dissolved in 3 .mu.L of formaldehyde/50 mM EDTA. The
resulting samples were loaded on wells in volumes of 2 .mu.L per
well for sequencing in ABI sequencers.
[0184] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0185] The nucleotide sequences of the Sequence Listing as well as
the amino acid sequences deduced from them were used as query
sequences against databases such as GenBank, SwissProt, BLOCKS, and
Pima II. These databases, which contain previously identified and
annotated sequences, were searched for regions of homology
(similarity) using BLAST, which stands for Basic Local Alignment
Search Tool (Altschul (1993) supra, Altschul (1990) supra).
[0186] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms such as the one described in Smith et al.
(1992, Protein Eng. 5:35-51), incorporated herein by reference,
could have been used when dealing with primary sequence patterns
and secondary structure gap penalties. The sequences disclosed in
this application have lengths of at least 49 nucleotides, and no
more than 12% uncalled bases (where N is recorded rather than A, C,
G, or T).
[0187] The BLAST approach, as detailed in Karlin et al. (supra) and
incorporated herein by reference, searched for matches between a
query sequence and a database sequence. BLAST evaluated the
statistical significance of any matches found, and reported only
those matches that satisfy the user-selected threshold of
significance. In this application, threshold was set at 10.sup.-25
for nucleotides and 10.sup.-14 for peptides.
[0188] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam); and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp) for homology. The relevant database for a
particular match were reported as GIxxx.+-.p (where xxx is pri,
rod, etc., and if present, p=peptide), as shown in Table 1. In
column 3 of Table 1, the product score is calculated as follows:
the % nucleotide or amino acid identity [between the query and
reference sequences] in BLAST is multiplied by the % maximum
possible BLAST score [based on the lengths of query and reference
sequences] and then divided by 100. Where an Incyte Clone was
homologous to several sequences, up to five matches were provided
with their relevant scores. In an analogy to the hybridization
procedures used in the laboratory, the electronic stringency for an
exact match was set at 70, and the conservative lower limit for an
exact match was set at approximately 40 (with 1-2% error due to
uncalled bases). Column 4 provides log-likelihood where the
value=log (probability.div.threshold); column 5--the relevant
GenBank release; and column 6--a GenBank description of the
protein, or an edited version thereof. Some of the GenBank
descriptions presented in the tables of this application were
standardized with respect to abbreviations and spelling.
[0189] Each cDNA was compared to sequences in GenBank using a
search algorithm developed by Applied Biosystems and incorporated
into the INHERIT 670 sequence analysis system. In this algorithm,
Pattern Specification Language (TRW Inc, Los Angeles, Calif.) was
used to determine regions of homology. The three parameters that
determine how the sequence comparisons run were window size, window
offset, and error tolerance. Using a combination of these three
parameters, the DNA database was searched for sequences containing
regions of homology to the query sequence, and the appropriate
sequences were scored with an initial value. Subsequently, these
homologous regions were examined using dot matrix homology plots to
distinguish regions of homology from chance matches. Smith-Waterman
alignments were used to display the results of the homology
search.
[0190] Peptide and protein sequence homologies were ascertained
using the INHERIT- 670 sequence analysis system using the methods
similar to those used in DNA sequence homologies. Pattern
Specification Language and parameter windows were used to search
protein databases for sequences containing regions of homology
which were scored with an initial value. Dot-matrix homology plots
were examined to distinguish regions of significant homology from
chance matches.
[0191] BLAST, which stands for Basic Local Alignment Search Tool
(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al.
(1990) J. Mol. Biol. 215:403-410), was used to search for local
sequence alignments. BLAST produces alignments of both nucleotide
and amino acid sequences to determine sequence similarity. Because
of the local nature of the alignments, BLAST is especially useful
in determining exact matches or in identifying homologs. BLAST is
useful for matches which do not contain gaps. The fundamental unit
of BLAST algorithm output is the High-scoring Segment Pair
(HSP).
[0192] An HSP consists of two sequence fragments of arbitrary but
equal lengths whose alignment is locally maximal and for which the
alignment score meets or exceeds a threshold or cutoff score set by
the user. The BLAST approach is to look for HSPs between a query
sequence and a database sequence, to evaluate the statistical
significance of any matches found, and to report only those matches
which satisfy the user-selected threshold of significance. The
parameter E establishes the statistically significant threshold for
reporting database sequence matches. E is interpreted as the upper
bound of the expected frequency of chance occurrence of an HSP (or
set of HSPs) within the context of the entire database search. Any
database sequence whose match satisfies E is reported in the
program output.
[0193] IV. Northern Analysis
[0194] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound
(Sambrook et al., supra).
[0195] Analogous computer techniques using BLAST (Altschul, S. F.
1993 and 1990, supra) are used to search for identical or related
molecules in nucleotide databases such as GenBank or the LIFESEQ
database (Incyte Pharmaceuticals). This analysis is much faster
than multiple, membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or
homologous.
[0196] The basis of the search is the product score which is
defined as: 1 %sequenceidentity .times. %maximumBLASTscore 100
[0197] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1-2% error; and at 70, the match will be exact.
Homologous molecules are usually identified by selecting those
which show product scores between 15 and 40, although lower scores
may identify related molecules.
[0198] The results of northern analysis are reported as a list of
libraries in which the transcript encoding hLC3 occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0199] V. Extension of hLC3-Encoding Polynucleotides to Full Length
or to Recover Regulatory Sequences
[0200] Full length hLC3-encoding nucleic acid sequence (SEQ ID
NO:2) is used to design oligonucleotide primers for extending a
partial nucleotide sequence to full length or for obtaining 5' or
3', intron or other control sequences from genomic libraries. One
primer is synthesized to initiate extension in the antisense
direction (XLR) and the other is synthesized to extend sequence in
the sense direction (XLF). Primers are used to facilitate the
extension of the known sequence "outward" generating amplicons
containing new, unknown nucleotide sequence for the region of
interest. The initial primers are designed from the cDNA using
OLIGO 4.06 (National Biosciences), or another appropriate program,
to be 22-30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures about
68.degree.-72.degree. C. Any stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations is
avoided.
[0201] The original, selected cDNA libraries, or a human genomic
library are used to extend the sequence; the latter is most useful
to obtain 5' upstream regions. If more extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0202] By following the instructions for the XL-PCR kit (Perkin
Elmer) and thoroughly mixing the enzyme and reaction mix, high
fidelity amplification is obtained. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR is performed using the Peltier Thermal Cycler
(PTC200; M. J. Research, Watertown, Mass.) and the following
parameters:
2 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat step 4-6 for 15 additional
cycles Step 8 94.degree. C. for 15 sec Step 9 65.degree. C. for 1
min Step 10 68.degree. C. for 7:15 min Step 11 Repeat step 8-10 for
12 cycles Step 12 72.degree. C. for 8 min Step 13 4.degree. C. (and
holding)
[0203] A 5-10 .mu.l aliquot of the reaction mixture is analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
the sequence. Bands thought to contain the largest products are
selected and removed from the gel. Further purification involves
using a commercial gel extraction method such as QIAQUICK (QIAGEN
Inc., Chatsworth, Calif.). After recovery of the DNA, Klenow enzyme
is used to trim single-stranded, nucleotide overhangs creating
blunt ends which facilitate religation and cloning.
[0204] After ethanol precipitation, the products are redissolved in
13 .mu.l of ligation buffer, l .mu.l T4-DNA ligase (15 units) and 1
.mu.l T4 polynucleotide kinase are added, and the mixture is
incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) are transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook et al., supra). After
incubation for one hour at 37.degree. C., the whole transformation
mixture is plated on Luria Bertani (LB)-agar (Sambrook et al.,
supra) containing 2.times.Carb. The following day, several colonies
are randomly picked from each plate and cultured in 150 .mu.l of
liquid LB/2.times.Carb medium placed in an individual well of an
appropriate, commercially-available, sterile 96-well microtiter
plate. The following day, 5,.mu.l of each overnight culture is
transferred into a non-sterile 96-well plate and after dilution
1:10 with water, 5 .mu.l of each sample is transferred into a PCR
array.
[0205] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction are added to each well. Amplification is
performed using the following conditions:
3 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0206] Aliquots of the PCR reactions are run on agarose gels
together with molecular weight markers. The sizes of the PCR
products are compared to the original partial cDNAs, and
appropriate clones are selected, ligated into plasmid, and
sequenced.
[0207] VI. Labeling and Use of Hybridization Probes
[0208] Hybridization probes derived from SEQ ID NO:2 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base-pairs, is
specifically described, essentially the same procedure is used with
larger cDNA fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
.mu.Ci of [.gamma.-.sup.32 P] adenosine triphosphate (Amersham) and
T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeled
oligonucleotides are substantially purified with Sephadex G-25
superfine resin column (Pharmacia & Upjohn). A portion
containing 10.sup.7 counts per minute of each of the sense and
antisense oligonucleotides is used in a typical membrane based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba I,
or Pvu II; DuPont NEN).
[0209] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times. saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film
(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimager
cassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,
hybridization patterns are compared visually.
[0210] VII. Antisense Molecules
[0211] Antisense molecules to the hLC3-encoding sequence, or any
part thereof, is used to inhibit in vivo or in vitro expression of
naturally occurring hLC3. Although use of antisense
oligonucleotides, comprising about 20 base-pairs, is specifically
described, essentially the same procedure is used with larger cDNA
fragments. An oligonucleotide based on the coding sequences of
hLC3, as shown in FIG. 1, is used to inhibit expression of
naturally occurring hLC3. The complementary oligonucleotide is
designed from the most unique 5' sequence as shown in FIG. 1 and
used either to inhibit transcription by preventing promoter binding
to the upstream nontranslated sequence or translation of an
hLC3-encoding transcript by preventing the ribosome from binding.
Using an appropriate portion of the signal and 5' sequence of SEQ
ID NO:2, an effective antisense oligonucleotide includes any 15-20
nucleotides spanning the region which translates into the signal or
5' coding sequence of the polypeptide as shown in FIG. 1.
[0212] VIII. Expression of hLC3
[0213] Expression of hLC3 is accomplished by subcloning the cDNAs
into appropriate vectors and transforming the vectors into host
cells. In this case, the cloning vector, PSPORT, previously used
for the generation of the cDNA library is used to express hLC3 in
E. coli. Upstream of the cloning site, this vector contains a
promoter for .beta.-galactosidase, followed by sequence containing
the amino-terminal Met, and the subsequent seven residues of
.beta.-galactosidase. Immediately following these eight residues is
a bacteriophage promoter useful for transcription and a linker
containing a number of unique restriction sites.
[0214] Induction of an isolated, transformed bacterial strain with
IPTG using standard methods produces a fusion protein which
consists of the first eight residues of .beta.-galactosidase, about
5 to 15 residues of linker, and the full length protein. The signal
residues direct the secretion of hLC3 into the bacterial growth
media which can be used directly in the following assay for
activity.
[0215] IX. Demonstration of hLC3 Activity
[0216] Microtubules are purified from adult rat brain by reversible
assembly (Vallee, R. B. (1982) Methods Enzymol. 134:89-104) or the
taxol method (Vallee, R. B. (1982) J. Cell Biol. 92:435-442) using
PEM buffer (100 mM PIPES, pH 6.6, 1 mM EGTA, 1 mM MgSO.sub.4). To
separate the MAPs from tubulin, the pellets from twice-cycled
microbubules are resuspended in PEM buffer and applied to a 0.1 M
MgSO.sub.4-saturated phosphocellulose column as described by
Sloboda, R. D. and Rosenbaum, J. L. ((1982) Methods Enzymol.
85:409-416). The fractions containing protein are applied to a
second phosphocellulose column.
[0217] In a total volume of 100 .mu.l, 20 .mu.l of hLC3 (250
.mu.g/ml) is added to 80 ml of whole microtubules (450 .mu.g/ml) or
tubulin (300 .mu.g/ml) and incubated at 37.degree. C. for 10
minutes in the presence of 1 mM GTP and 50 .mu.M taxol. The
suspension is centrifuged, the supernatant is removed, and the
microtubule pellet is resuspended to the original reaction volume
in PEM buffer. To assess the partitioning of LC3 between the
supernatant and pellet fractions, equal amounts of supernatant and
resuspended pellet are placed in SDS sample buffer and assayed on a
5-20% gradient SDS polyacrylamide gel stained with Coomassie
Brilliant Blue.
[0218] X. Production of hLC3 Specific Antibodies
[0219] hLC3 that is substantially purified using PAGE
electrophoresis (Sambrook, supra), or other purification
techniques, is used to immunize rabbits and to produce antibodies
using standard protocols. The amino acid sequence deduced from SEQ
ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc.) to
determine regions of high immunogenicity and a corresponding
oligopolypeptide is synthesized and used to raise antibodies by
means known to those of skill in the art. Selection of appropriate
epitopes, such as those near the C-terminus or in hydrophilic
regions, is described by Ausubel et al. (supra), and others.
[0220] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems Peptide Synthesizer Model
431A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma, St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,
supra). Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. The resulting antisera are tested for
antipeptide activity, for example, by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radioiodinated, goat anti-rabbit
IgG.
[0221] XI. Purification of Naturally Occurring hLC3 Using Specific
Antibodies
[0222] Naturally occurring or recombinant hLC3 is substantially
purified by immunoaffinity chromatography using antibodies specific
for hLC3. An immunoaffinity column is constructed by covalently
coupling hLC3 antibody to an activated chromatographic resin, such
as CnBr-activated Sepharose (Pharmacia & Upjohn). After the
coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0223] Media containing hLC3 is passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of hLC3 (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/hLC3 binding (eg, a buffer of pH
2-3 or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and hLC3 is collected.
[0224] XII. Identification of Molecules Which Interact with
hLC3
[0225] hLC3 or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent (Bolton, A. E. and W. M.
Hunter (1973) Biochem. J. 133:529-539). Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated
with the labeled hLC3, washed and any wells with labeled hLC3
complex are assayed. Data obtained using different concentrations
of hLC3 are used to calculate values for the number, affinity, and
association of hLC3 with the candidate molecules.
[0226] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
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